autoantibodies and autoantibody-negative children (IV)
Previous studies have indicated that factors affecting the gut, such as altera- tions in the intestinal microbiota, are capable of modulating the development of autoimmune diabetes. Several animal studies have provided information regard- ing the influence of the intestinal microbiota on the development of autoimmune diabetes. The gut microbiota differs between biobreeding diabetes-prone (BB- DP) and diabetes resistant (DR) rats. Stool samples from BB-DR rats studied before the onset of diabetes contained higher populations of Lactobacillus and
Bifidobacterium, whereas BB-DP rats had higher number of Bacteroides, Eu- bacterium, and Ruminococcus (Roesch et al. 2009). Antibiotics, which modulate
gut microbiota, can also prevent autoimmune diabetes in BB-DP rats. In a non- obese diabetic mouse model (NOD), the pathogen-free mice lacking MyD88, a TLR signaling molecule, did not develop diabetes, which emphasizes the role of the intestinal microbiota as a regulator of autoimmune diabetes (Wen et al. 2008). The aim of this study was to compare the composition of the gut micro- biota between children with β-cell autoimmunity and autoantibody-negative children matched for age, gender, HLA risk genotype, and early feeding history.
In our study, the analysis of all samples at the phylum level showed that
Firmicutes (58.1%), Actinobacteria (36.2%), and Bacteroidetes (3.4%) were the
dominant phyla. The most common families were the Bifidobacteriaceae (32.8%) (Actinobacteria), Lachnospiraceae (18.4%) (Firmicutes), and Rumino-
coccaceae (17.1%) (Firmicutes). Bifidobacterium was the most frequent genus
(34.2%). There were differences in the composition of intestinal microbiota between the autoantibody-positive and autoantibody-negative children. In
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autoantibody-positive children, the phylum Bacteroidetes, family Bacteroi-
daceae (2.5%) and the genus Bacteroides (3.1%) were more common than in
autoantibody-negative children (p < 0.05, p <0.05, and p <0.05, respectively). There have only been a few previous studies on the intestinal microbiota in rela- tion to T1D in humans. However, in accordance with our results, an earlier fol- low-up study including four case-control pairs suggested that the ratio of Bac-
teroidetes to Firmicutes increased over time in those children who eventually
progressed to clinical T1D, while it decreased in children who remained non- diabetic (Giongo et al. 2011). A role of the genus Bacteroides in the develop- ment of autoimmune diabetes has been implicated in both animal models and humans (Brown et al. 2011).
Principal component analysis (PCA) on the species level showed various correlations with β-cell autoimmunity. The first principal component (PC) corre- lated positively with Bifidobacterium adolescentis (11%), Faecalibacterium
prausnitzii (5.6%), Clostridium clostridioforme (2.3%), and Roseburia faecis
(0.94%), which are considered as short-chain fatty acid-producing species. The abundance of lactate and butyrate-producing bacteria was also inversely related to the number of β-cell autoantibodies in children; the lowest levels were ob- served in children positive for three or four autoantibodies (original publication IV, Figure 1).
The second and the third PC were strongly related to the abundance of both
B. adolescentis and Bifidobacterium pseudocatenulatum (original publication
IV, Figure 2). B. adolescentis was the most common species (15.8%) amongst the children in the TRIGR pilot study (older group), whereas B. pseudocatenula-
tum (younger group) was most frequent (15.8%) in the children from the FIN-
DIA study. Samples from children with autoantibody-positivity from both age groups were overrepresented, in which the combined abundance of B. adoles-
centis and B. pseudocatenulatum was below 12% (see Figure 2 in original publi-
cation IV).
At level of single bacterial species, Roseburia faecis (0.94%) was present in greater proportions in autoantibody-negative than -positive children, whereas the butyrate-producing bacterium Eubacterium hallii (6.0%) inversely correlated with the total number of autoantibodies. Bifidobacterium animalis (0.18%),
Lactobacillus acidophilus (0.03%), and Clostridium perfringens (0.03%) were
more abundant in children with β-cell autoimmunity than in those without. Butyrate is thought to be beneficial, as it is the main energy source for colo- nic epithelial cells (Hague et al. 1996). Butyrate-producing bacteria represent a functional group within the microbial community of the human large intestine. Despite their heterogeneity, butyrate plays a key role in maintaining human gut health, as the major source of energy to the colonic mucosa, and as an important regulator of gene expression, inflammation, differentiation, and apoptosis in host cells (Louis and Flint 2009). Furthermore, butyrate has been shown to regulate
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the assembly of tight junctions and gut permeability (Peng et al. 2009). In our study, Roseburia faecis, which produces butyrate using acetate (Duncan et al. 2002), showed the most apparent inverse relation with autoantibody positivity. In addition, E. hallii (6.0%), which produces butyrate from lactate and acetate, was inversely correlated with the numbers of autoantibodies. A recent study including four pairs of cases who developed T1D and autoantibody-negative controls suggested that higher proportions of butyrate-producing and mucin- degrading bacteria were observed in autoantibody-negative subjects than in T1D cases (Brown et al. 2011).
The administration of butyrate has been tested in the prevention of autoim- mune diabetes in a BB rat model. Although the enteral administration of sodium butyrate shortly after birth during the weaning period in BBDP rats did not re- duce the subsequent development of autoimmune diabetes, it modulated the gut inflammatory response (Li et al. 2010).
In this cohort, the total duration of breastfeeding, but not the duration of ex- clusive breastfeeding, showed an inverse association with autoantibody positiv- ity. The duration of breastfeeding correlated positively with the abundance of B.
adolescentis and Blautia hansenii. Coprococcus comes were more common in
children who received CM formula before the age of 6 months than in exclu- sively breast-fed children. The abundance of the genus Bifidobacterium corre- lated with the total duration of breastfeeding although the children in the TRIGR pilot study were studied several years later. This is in accordance with previous reports that indicated the long-lasting effects of breastfeeding on the intestinal microbiota in children (Harmsen et al. 2000).
Interestingly, B. animalis and L. acidophilus were found to be extremely ab- undant in autoantibody-positive children who had received a hydrolyzed whey- based infant formula, whereas their corresponding controls, who had received the same infant formula, did not have a high abundance of either.
The diversity of the intestinal microbiota was significantly higher in the chil- dren in the TRIGR pilot cohort (older age group) than in the children in the FINDIA pilot cohort (younger age group). Furthermore, there was a trend to- wards a higher diversity per sample in the autoantibody-negative than the autoantibody-positive children, especially in the children from the TRIGR pilot study. This is in agreement with an earlier study showing that microbial diver- sity decreased with increasing age in the four children who developed T1D and was lower in autoantibody-positive than in autoantibody-negative children (Giongo et al. 2011).
It is known that the 16S method in studies provides information on the num- ber of known species in the microbiome, but no information is provided on the function of these bacteria. Moreover, the methods whereby intestinal bacterial samples are collected have an affect on the results and must be standardized. Thus, larger cohort studies are needed to further define alterations in the compo-
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sition of the intestinal microbiome in humans and the underlying mechanisms that lead to autoimmunity and diabetes development.
To conclude, our study indicated the importance of bifidobacteria and lactate and/or butyrate-producing species in relation to the development of β-cell auto- immunity. Bifidobacteria not only supply butyrate-producing species with lac- tate and acetate, but they also enhance the intestinal epithelial barrier function by modulating the gut mucosa. In children with β-cell autoimmunity, the higher proportion of bacteria of the phylum Bacteroides was confirmed. The findings demonstrate specific changes in the composition of intestinal bacteria in children with β-cell autoimmunity, and suggest that dysbiosis is seen at the prediabetic stage of the disease.
The development of immune responses
6 CONCLUSIONS
I. In healthy infants with a T1D-associated HLA-mediated risk, altered T cell differentiation was observed in cord blood-derived cells. The expression of tran- scription factor GATA-3 and chemokine receptor CCR4 was reduced in the Th2 environment in children at risk. This suggests that infants with a T1D-associated risk genotype may develop altered immune responses to environmental factors, and this could be associated with the risk of T1D.
II. Enchanced levels of antibodies to CM proteins were observed in infancy in those children who later progressed to T1D when compared with children who remained healthy. The results indicate that children who progress to autoim- mune diabetes have increased antibody responses to the cow’s milk proteins during the first year of life. This may reflect a dysfunctional gut immune system in early infancy. The results suggest that dysregulation of oral tolerance is pre- sent years before the actual onset of the disease in children who later progress to T1D.
III. We demonstrated that the expression of transcription factor FOXP3 in cord blood regulatory T cells was higher in the offspring of mothers with T1D when compared with the infants of unaffected mothers. After in vitro insulin stimula- tion, the expression of FOXP3 in CD4+CD25+ regulatory cells, and up- regulation of transcription factors (FOXP3, IL-10, TGF-beta, NFATc2, and STIM1) were only increased in the offspring of mothers with T1D. The results suggest that maternal insulin therapy specifically increases regulatory T-cell activation and triggers the development of tolerance to insulin in the fetus. This may explain the lower risk of diabetes in children with maternal vs. paternal diabetes.
IV. Principal component analysis indicated that a low abundance of lactate- and butyrate-producing species was associated with β-cell autoimmunity. Further- more, a low abundance of two Bifidobacterium species, B. adolescentis and B.
pseudocatenulatum, but an increased abundance of the Bacteroides genus was
observed in the children with β-cell autoimmunity. The low abundance of bifi- dobacteria and butyrate-producing species could adversely affect the intestinal epithelial barrier function and inflammation, whereas the apparent importance of the genus Bacteroides in T1D, possibly as an immunomodulator, is insuffi- ciently understood.
The development of immune responses
7 ACKNOWLEDGEMENTS
The present work was carried out over the years 2003–2012 at the Immune Re- sponse Unit, the National Institute for Health and Welfare, and Children’s Hos- pital, University of Helsinki.
I am deeply grateful to all people who have contributed to this work. In particu- lar, I warmly thank:
Professor Pekka Puska, the Director of the National Institute for Health and Welfare, and Terhi Kilpi, the Head of the Department of Vaccines and Immune Protection, the National Institute of Health and Welfare, for providing excellent research facilities.
Docent Jari Petäjä (Director of the Department of Gynecology and Pediat- rics, Helsinki University Central Hospital), Professor Mikael Knip (Chair of the Children’s Hospital, University of Helsinki), and Docent Eero Jokinen (Head of the Department of Pediatrics, Helsinki University Central Hospital) for provid- ing excellent facilities to carry out this study.
Professor Markku Heikinheimo, Head of the Institute of Clinical Medicine and former Head of the National Graduate School of Clinical Investigation, and former Head of the Pediatric Graduate School at University of Helsinki, and Docent Jussi Merenmies, Head of the Pediatric Graduate School, for their valu- able work in bringing together and supporting young investigators.
All the children and their families, for participating in these long-lasting stu- dies.
Professor Outi Vaarala, my supervisor, for teaching me the principles of the scientific research, sharing her knowledge with me in the field of diabetes im- munology, friendship, and support at various stages of this work.
Emeritus Professor Hans Åkerblom, the first Principal Investigator of the Trial to Reduce IDDM in Genetically at Risk, my supervisor, for the opportunity to participate in the diabetes intervention study and continuous encouragement. His lifework dedication to diabetes research has made my work possible.
Docent Aaro Miettinen and Docent Jussi Kantele, for their thorough review of the thesis and for providing constructive comments.
Emeritus Professor Erkki Savilahti and Docent Jukka Rajantie, for their valuable advice and sincere support as members of the follow-up group in the Pediatric Graduate School.
All the co-authors, Professor Mikael Knip for his encouraging attitude to- wards scientific work and never-ending enthusiasm, Professor Jorma Ilonen for providing excpertise in genetics during these years, Professor Suvi Virtanen, Professor Gjalt Welling, Professor Hermie Harmsen, Taina Härkönen, Susanne
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Skarsvik, Marcus de Coffau, Terhi Ruohtula, Janne Nieminen, Laura Orivuori, and Saara Hakala, for valuable contribution to this study.
Anneli Suomela, Harry Lybeck, Maria Kiikeri, Sinikka Tsupari, and Pirjo Mäki, for their skilful technical assistance, and Tarja Alander, for her adminis- trative assistance in the Immune Response Unit.
All my former and present colleagues in our lab, Jarno Honkanen, for inspir- ing discussions and support during these years, Terhi Ruohtula, for great times shared at work and on several conference trips, Minna Tiittanen, Paula Klemetti, Johanna Paronen, Heli Siljander, Riikka Nissinen, Harri Salo, Emma Marschan, Emma Savilahti, Saara Aittomäki, Janne Nieminen, Veera Hölttä, Linnea Hart- wall, Laura Orivuori, and Monika Paasela, for their support and companionship in and out of lab.
Marja Salonen, Hilkka Puttonen, Tarja Tenkula, Kristiina Merentie, Heli Suomalainen, Katriina Koski, Matti Koski, Mila Hyytinen, Eeva Pajakkala and Päivi Kleemola, for their excellent collaboration in the TRIGR Study. To work and travel with you over the years has been a great pleasure.
Sirpa Nolvi and Anne Björk, for their excellent collaboration.
All the doctors, nurses and other personnels of the TRIGR study in Finland, for pleasant collaboration, and the personnel of the Research Laboratory of Children’s Hospital, for skilfull assistance.
My friends outside research, especially Marjaana and her family, for their friendship.
My dear parents, Marja and Aarne, and godmothers, Anja and Eine, for their endless support throughout my life, and my brother Matti, and his family, for their friendship and encouragement. I wish my mother could have been here today to see me reach this goal.
Finally, with all my heart, I want to thank my children Anni and Olli, for their love, and inspiration, and Juha, without your love, unfailing support and patience through the years, this work would never have been completed.
This study was financially supported by the National Graduate School of Clinical Investigation, the Päivikki and Sakari Sohlberg Foundation, the Finnish Medical Society Duodecim, the Finnish Diabetes Foundation and Finska Läka- resällskapet.
Helsinki, October 2012 Kristiina Luopajärvi
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